Proteomics

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A Proteome-Wide Map of Chaperone-Assisted Protein Refolding in a Cytosol-like Milieu


ABSTRACT: The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chain’s intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting proteins safe passage to their native states; however, the complexity of this medium has generally precluded biophysical techniques from interrogating protein folding under cellular-like conditions for single proteins, let alone entire proteomes. Here, we develop a limited-proteolysis mass spectrometry approach paired within an isotope-labeling strategy to globally monitor the structures of refolding E. coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data, and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than indirect measures like binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones both unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. The data support a model in which chaperone-nonrefolders have evolved to fold efficiently once and only once, co-translationally, and remain kinetically trapped in their native conformations.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Escherichia Coli

SUBMITTER: Stephen Fried  

LAB HEAD: Stephen Fried

PROVIDER: PXD030869 | Pride | 2022-11-23

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
20210524_CD_N1.raw Raw
20210524_CD_N2.raw Raw
20210524_CD_N3.raw Raw
20210524_CD_R1min.pdResult Other
20210524_CD_R1min_1.raw Raw
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